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Fig. 1. Expression of RyR in human DCs. Peripheral blood monocytes were induced to differentiate into iDCs by 5-day culture in IL4 and GM-CSF. (A) Cells were then washed and incubated in the presence of fluorochrome-labeled monoclonal antibodies (mAbs) recognizing the indicated surface markers. Specific fluorescence (full histograms) was evaluated by taking advantage of a FACSCalibur flow cytometer equipped with Cell Quest software (Becton Dickinson) using, as negative controls, isotype-matched irrelevant reagents (empty histograms). One representative experiment out of five is shown. (B) Total RNA was extracted from immature (iDCs), LPS-matured (mature DCs) and from other cell types, and the expression of genes encoding the different RYR isoforms was evaluated by RT-PCR. RNA from EBV-transformed lymphocytes was used as control for RYR1 isoform gene expression. (C) Immunofluorescence analysis of iDCs. Cells were stained with goat anti-RyR polyclonal Ab followed by Alexa Fluor-555-conjugated anti-goat Ab (a-j), Alexa Fluor-555-conjugated anti-goat Ab alone (k) or with allophycocyanine-conjugated anti-CD1a (l). Immunofluorescence analysis on acetone:methanol-fixed iDCs reveals strong perinuclear RyR fluorescence that extends into the endoplasmic reticulum network. Panels a-j show 1 µm optical slices from the bottom upwards; bar, 10 µm. Images were acquired with a 100x Plan Neofluar oil immersion objective (NA 1.3) mounted on a Zeiss Axiovert 100 confocal microscope. Panel l: paraformaldehyde fixed DCs display typical plasma membrane fluorescence for the CD1a marker (bar, 20 µm).
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